Laboratory reactivity of serum samples from patients with certain diseases can often be very similar. This is mainly due to the antigenic sharing among agents related to the etiology of such diseases. In these cases, conventional serological methods of diagnosis have as main disadvantage the low specificity, contributing to false-positive results and difficulties in differential and confirmatory serologic diagnosis of the disease.
For example, the laboratory diagnosis of Chagas' disease, especially in the chronic phase of infection, based on the use of serological methods for detection of anti-T. cruzi. According to the Brazilian Consensus on Chagas Disease (Brazilian Consensus in Chagas Disease—Surveillance Department of the Ministry of Health, Journal of the Brazilian Society of Tropical Medicine VOL. 38: SUPPLEMENT III, 2005), serologic tests of choice for diagnosing the disease are: Indirect Hemagglutination (IHA), the Indirect Immunofluorescence Assay (IFA) and Enzyme-Linked Immunosorbent Assay (ELISA). These tests, known as conventional, are commonly used in the diagnosis of Chagas' disease in clinical laboratories and serological screening in blood banks. When applied together, they show high sensitivity, however, due to the large antigenic sharing among parasites belonging to the Tripanosomatidae family, as is the case of Leishmania spp.; conventional serological methods of diagnosis have as main disadvantage the low specificity, contributing to false-positive results and difficulties in differential and confirmatory serologic diagnosis of the Chagas' disease and leishmaniasis (Marchi et al, 2007). This issue gains even greater proportions when designed in the reality of co-endemic regions for Chagas' disease and leishmaniasis, which is reaching global dimensions as a result of the processes of population migration and environmental changes.
The application of two tests of distinct principles for the diagnosis of T. cruzi infection in Blood Banks, as recommended by the Brazilian Ministry of Health, allowed the desired decrease in transmission of Chagas' disease by blood transfusion; on the other hand, a new problem arose, associated with the increase in the number of patients with inconclusive or non-negative serological results. Thus, a major problem in serological screening for Chagas' disease in blood donors is the high frequency of non-negative or indeterminate reactions, which causes many healthy subjects to be stained as suffering from a serious illness, and promotes significant unnecessary discarding of blood units in blood banks and major financial losses for the health system.
Searching the scientific literature, one can notice the difficulty of eliminating cross-reactivity in immunofluorescence analysis by flow cytometry of trypanosomatids when this technique is applied to the diagnosis of T. cruzi infection or for the diagnosis of some species of Leishmania sp., and there is no description of diagnostic of the three diseases simultaneously by the method of immunofluorescence by flow cytometry.
Thus, there is a clear need for a technical solution that allows the use of flow cytometry, a technique with high sensitivity and specificity for the simultaneous diagnosis of pathologies in which the specific antibodies to be detected are directed at targets, or biological agents, morphologically similar.
The literature points to “Luminex” technology (Bonetta, L., Flow Cytometry smaller and better. Nature Methods 2(10): 785-795, 2005), that also uses a system of incremental labeling with fluorochromes as a facilitator to conduct multiple analyzes on a single platform of reaction for immunofluorescence by flow cytometry. However, the LUMINEX system differs from the triplex method for using microspheres incrementally stained with fluorochromes bound to antibodies or antigens, not aiming at the incremental labeling directly in the target biological agent of antibody reactivity, such as the Triplex method does. Furthermore, there are no assays using the Luminex method, using fluorescent microspheres bound to antigenic constituents derived from pathogens, specifically excreted or secreted antigens (ES-antigens), as well as selective antigens of plasma membrane of pathogens free of contamination with antigens derived from other cellular compartments, which can be achieved with the Triplex method. In addition, in the invention described herein the labeling can be carried out directly using the full parasite, such that the antigenic diversity available to assist in the detection is increased.
The triplex method also has the potential of incorporating the “multiplex” mode with the insertion of new molecular targets, then allowing the simultaneous detection of IgG1 reactivity against other pathogens, such as Tachyzoites of Toxoplasma gondii, spirochete of Treponema pallidum and even target cells infected with HTLV. These represent a practical application of the triplex method in screening assays in blood banks.
The high number of inconclusive reactions reinforces the real and urgent need to develop more specific serological tests or practical and rapid confirmatory tests, which can be introduced in clinical laboratories routine and blood bank services, reducing unnecessary disposal of blood bags and the difficulty in conducting donors with indeterminate reactions.